There are over nine million dairy cows in the United States, over one million in Canada and over fifty million worldwide. The dairy industry is extremely competitive and the ability of a dairy to maintain pregnancies post insemination is critical to the profitability of the producer. It is estimated that the cost of a non-pregnant cow is about five dollars per day. It is further estimated that current inseminations result in approximately thirty to forty percent pregnant cows at day 45 and of those cows ninety to ninety-five percent deliver calves at the end of the 283-day gestation period. Reproductive efficiency in dairy cattle has been declining steadily over a prolonged period of time. The magnitude and the consistency of this trend are of great importance to the dairy industry and amount to a steady decline of approximately one percent in first service conception rates per year for the last ten years. The impact of this change in productivity has not been readily apparent, because individual cow milk production has increased by twenty percent over the same period. In the long run, the dairy industry cannot afford to continue the current rate of declining reproductive performance.
The primary revenue source in the dairy industry is milk production. Progress in genetics and management of dairy cows has led to remarkable increases in milk production throughout the last several decades, with a twenty percent increase in per-cow production in the last ten years alone (USDA National Agricultural Statistics Service, http//www.usda.gov/nass). In order to maintain high herd productivity, however, cows must become pregnant and deliver a calf so that the lactation cycle is renewed. Additionally, sufficient numbers of heifers must be produced to replace older cows. Therefore, the future productivity of the dairy industry is very dependent on the maintenance of fertility and reproduction.
During the same time that milk production per cow has increased, however, reproductive efficiency of dairy cows has steadily declined. Colorado dairy herds are among the most productive in the nation, and the state currently ranks second in average yearly per-cow production (USDA National Agricultural Statistics Service, http//www.usda.gov/nass). The Colorado dairy industry is typical for the national trend in declining cow fertility. From 1992 through the present, while milk production has increased from 21,000 to 24,000 lbs/cow/yr, average days open (days until conception) have increased from 130 to 173 days. The first service conception rate for Colorado herds has declined from 51% to 37%, and the rate of services per conception has risen from 2 to 2.8 during the last 10 years (data from Dairy Herd Improvement Association, www.dhiprovo.com).
Declining reproductive efficiency of dairy cattle has been observed throughout the United States, and other parts of the world where milk production has been increasing (Lucy, M. C., “Reproductive loss in high-producing dairy cattle: Where will it end?,” J. Dairy Sci., 84:1277-1293, 2001; Roche, J. F. et al., “Reproductive management of postpartum cows,” Anim. Reprod. Sci., 60-61:703-712, 2000; Royal, M. D. et al., “Declining fertility in dairy cattle: changes in traditional and endocrine parameters of fertility,” Anim. Sci., 70:487-502, 2000; and Macmillan, K. L. et al., “The effects of lactation on the fertility of dairy cows” Aust. Vet. J, 73:141-147, 1996). The strong temporal association between increasing milk production and decreasing fertility does not provide a known cause of the phenomenon. While the relationship between milk production and fertility appears to be antagonistic (Dematawewa, C. M., and P. J. Berger, “Genetic and phenotypic parameters for 305 day yield, fertility, and survival in Holsteins,” J. Dairy Sci., 81:2700-2709, 1998; and Hansen, L. B., “Consequences of selection for milk yield from a geneticist's viewpoint,” J. Dairy Sci., 83:1145-1150, 2000), some studies demonstrate a neutral effect of milk production per se (Grohn, Y. T. and P. J. Rajala-Schultz, “Epidemiology of reproductive performance in dairy cows,” Anim. Repro. Sci., 60-61:605-614, 2000), and other studies show higher producing herds have better reproductive performance than lower producing herds (Nebel, R. L. and M. L. McGilliard, “Interactions of high milk yield and reproductive performance in dairy cows,” J. Dairy Sci., 76:3257-3268, 1993; and Stevenson, J. S., “Can you have good reproduction and high milk yield?” Hoard's Dairyman, 145:202-203, 1999). Numerous features of high producing cows may negatively influence fertility, including negative energy balance and disease events such as retained placenta, ketosis, cystic ovary, and mastitis (Lucy 2001, supra; Grohn 2000, supra; and Staples, C. R. et al., “Relationship between ovarian activity and energy status during the early postpartum period of high producing dairy cows,” J. Dairy Sci., 73:938-947, 1990). A prominent trend in the U.S. dairy industry is decreased number of dairy farms, steadily increasing herd size, and movement of dairy production to the western states (USDA National Agricultural Statistics Service, http//www.usda.gov/nass). Larger herd size may contribute to decreased reproductive performance because of the associated changes in the dairy labor force and cow management, resulting in poorly trained or over tasked workers identifying estrus behavior, performing artificial insemination, conducting estrus synchronization programs, and identifying and treating sick cows (Lucy 2001, supra). Heat stress, which occurs throughout much of the year in western and southwestern US dairy herds, has significant negative impact on cattle fertility (Wolfenson, D. et al., “Impaired reproduction in heat-stressed cattle: basic and applied aspects,” Anim. Reprod. Sci., 60-61:535-547, 2000).
Recent studies with ultrasonic pregnancy detection demonstrate embryonic losses of at least 20% between 28 and 60 days of pregnancy (Pursley, J. R. et al., “Effect of time of artificial insemination on pregnancy rates, calving rates, pregnancy loss, and gender ratio after synchronization of ovulation in lactating dairy cows,” J. Dairy Sci., 81:2139-2144, 1998; and Vasconcelos, J. L., et al., “Pregnancy rate, pregnancy loss, and response to heat stress after AI at 2 different times from ovulation in dairy cows” Biol. Reprod., 56 (Supp. 1):140, 1997), and there are likely even higher losses prior to 28 days that are undetected by ultrasound examination (Lucy 2001, supra). Data suggest that modern dairy cows fail to establish pregnancy because of suboptimal uterine environment (Gustafsson, H. and K. Larsson, “Embryonic mortality in heifers after artificial insemination and embryo transfer: differences between virgin and repeat breeder heifers,” Res. Vet. Sci., 39:271-274, 1985). Although there are numerous possible factors that could be responsible for embryonic losses, one potential cause is low blood progesterone concentration.
Progesterone is required to maintain pregnancy in cattle, and low progesterone concentrations are associated with infertility. Blood progesterone concentrations are influenced by rates of secretion and metabolism/clearance. There is evidence that modern dairy cows maintain lower blood progesterone concentrations than those measured in cattle several decades ago (Lucy, M. C. et al., “Reproductive endocrinology of lactating dairy cows selected for increased milk production,” J. Anim. Sci., 76 (Suppl. 1):296, 1998). Larger corpora lutea secrete more progesterone and have a positive effect on pregnancy recognition and pregnancy rates, but there is evidence that dairy cows have smaller than desirable corpora lutea in some circumstances (Lucy 2001, supra; Vasconcelos, J. L. M. et al., “Reduction in size of the ovulatory follicle reduces subsequent luteal size and pregnancy rate,” Theriogenology, 56:307-314, 2001). The liver is the primary site of progesterone metabolism. Recent studies show that increased feed intake increases liver blood flow and increases the rate of progesterone clearance, thus decreasing serum progesterone concentrations (Sangsritavong, S. et al., “Liver blood flow and steroid metabolism are increased by both acute feeding and hypertrophy of the digestive tract,” J. Anim. Sci., 78(Suppl 1)221, 2000; and Wiltbank, M. C. et al., “Novel effects of nutrition on reproduction in lactating dairy cows,” J. Dairy Sci., 84(Suppl. 1):84, 2001).
Low serum progesterone during the luteal phase of the estrus cycle would be associated with low first service conception rate. Low progesterone concentrations may result from inadequate secretion, or alternatively high levels of metabolism/clearance, even when insemination has produced a potentially viable embryo. Low progesterone would allow the generation of prostaglandin by uterine endometrium at around day 16 of the estrus cycle, resulting in luteolysis and induction of ovulation, thus embryonic death and failure to maintain the pregnancy (Binelli, M. et al., “Antiluteolytic strategies to improve fertility in cattle,” Theriogenology, 56:1451-1463, 2001).
Binelli et al. 2001, supra, reviews antiluteolytic strategies for improving fertility in cattle.
In cows, the estrus cycle is about 21 days. To determine when a cycling cow is ready for breeding, the cow can be observed for behavioral estrus. Alternatively, a cow can be induced or forced into estrus with effective hormone therapies. Estrus of an entire herd can be synchronized (U.S. Pat. No. 3,892,855 issued Jul. 1, 1975, and U.S. Pat. No. 4,610,687 issued Sep. 9, 1986; U.S. Patent Application No. 60/380,042; Wilson, T. W., “Estrous Synchronization for Beef Cattle,” (June 2003), Bulletin 1232, the University of Georgia College of Agricultural and Environmental Sciences and the U.S. Department of Agriculture cooperating). Estrus synchronization, or preferably ovulation synchronization, is used in timed artificial insemination (TAI) breeding programs. TAI breeding programs involve precise estrus synchronization, which allows for timed breeding without monitoring for behavioral estrus. Examples of methods for forcing estrus include U.S. Pat. No. 5,589,457 (issued Dec. 31, 1996), Ovsynch (Pharmacia Animal Health, Peapack, N.J.), Cosynch, Select Synch, Modified Select Synch, MGA/PGF, and Syncro-Mate-B. Such methods typically employ hormones such as prostaglandins, e.g. PGF2α (Lutalyse®, Pharmacia Upjohn, Peapack, N.J.; Bovilene®, Syntex; Animal Health, Des Moines, Iowa; and Estrumate® Haver Lockhart, Shawnee, Kans.), and gonadotropin releasing hormone (GnRH). Ovsynch involves a GnRH injection followed by a prostaglandin injection one week later, followed by a second GNRH injection 48 hours later. Insemination is ideally then performed at 12-18 hours, preferably about 16 hours, after the second GNRH injection. Ovsynch is maximally effective when implemented between Days 18-20 of a 20-day bovine estrus cycle (Thatcher, W. W. et al. (2000) “New Strategies to Increase Pregnancy Rates” www.naab-css.org/education/Thatcher.html). Presynch (Pharmacia Animal Health, Peapack, N.J.) can be used to synchronize heifers before implementing Ovsynch. Ovsynch is maximally effective when implemented between Days 18-20 of a 20-day bovine estrus cycle (studies show starting between day 5-12 is best. (Moreira, F. et al. (2000) “Effect of day of the estrous cycle at the initiation of a timed insemination protocol on reproductive responses in dairy heifers,” J Anim Sci 78:1568-1576, 2000; Vasconcelos J L M, et al. (1999) “Synchronization rate, size of the ovulatory follicle, and pregnancy rate after synchronization of ovulation beginning on different days of the estrous cycle in lactating dairy cows,” Theriogenology 52: 1067-1078, 1999). Presynch involves two prostaglandin injections. Certain of the above-mentioned methods are also used on non-cycling cows to induce cycling, such as in lactating dairy cows. However, these protocols do not induce cyclicity—only progesterone priming does that. The only synchronization program that does induces cyclicity in lactating cows is using a controlled internal drug release device (CIDR) which releases progesterone or melengestrol acetate (MGA), which is illegal in lactating dairy cows). After precise estrus synchronization, animals need not be monitored for behavioral estrus and may be bred by appointment. Some animals may need estrus presynchronization before estrus synchronization. Melengestrol acetate (MGATM) in feed (Imwalle, D. B. et al. (1998) “Effects of melengestrol acetate on onset of puberty, follicular growth, and patterns of luteinizing hormone secretion in beef heifers” Biol. Repro. 58:1432-1436) or implants (U.S. Patent Publication No. 2001/0041697, published Nov. 15, 2001) can be used for presynchronizing estrus in heifers. Resynch is a program whereby animals are synchronized and bred, and then those animals that are determined to be open (not pregnant) are again synchronized and rebred.
Previous research has shown conflicting results on reproductive cycles and conception rates of cows receiving hCG (Eduvie and Seguin (1982) Theriogenology 17:415-422; Helmer and Britt (1986) Theriogenology 26:683-695; Sianangama and Rajamahendran (1992) Theriogenology 38:85-96; Diaz et al. (1998) J. Anim. Sci. 76:1929-1936; Sianangama and Rajamahendran (1996) Theriogenology 45:977-990; Schmitt et al. (1996) J. Anim. Sci. 74:1074-1083; and Schmitt et al. (1996) J. Anim. Sci. 74:1915-1929).
Thatcher et al. (2001 Theriogenology 55:75-89) describes the effects of hormonal treatments on the reproductive performance of cattle. Hormonal treatments include administration of bovine somatotrophin (bST) and hCG. D'Occhio et al. (2000 Anim. Reprod. Sci. 60-61:433-442) describes various strategies for beef cattle management using GNRH agonist implants. De Rensis et al. (2002 Theriogenology 58(9):1675-1687) describes the effect on dairy cows of administering GNRH or hCG before artificial insemination. Martinez et al. (1999 Anim. Reprod. Sci. 57:23-33) describes the ability of porcine luteinizing hormone (LH) and gonadotropin releasing hormone (GNRH) to induce follicular wave emergence in beef heifers on Days 3, 6, and 9 of the estrus cycle, after ovulation (Day 0), without insemination. Santos et al. (2001 J. Animal Science 79:2881-2894) describes the effect on reproductive performance of intramuscular administration of 3,300 IU of human chorionic gonadotropin (hCG) to high-producing dairy cows on Day 5 after artificial insemination. Lee et al. (1983 Am. J. Vet. Res. 44(11):2160-2163) describes the effect on dairy cows of administering GNRH at the time of artificial insemination (AI). U.S. Pat. No. 5,792,785 (issued Aug. 11, 1998) and U.S. Pat. No. 6,403,631 (issued Jun. 11, 2002) describe methods and compositions for administering melatonin before and after insemination to enhance pregnancy success in an animal. Chagas e Silva et al. (2002 Theriogenology 58(1):51-59) describes plasma progesterone profiles following embryo transfer in dairy cattle. Weems et al. (1998 Prostaglandins and other Lipid Mediators) describes the effects of hormones on the secretion of progesterone by corpora lutea (CL) from non-pregnant and pregnant cows. U.S. Pat. No. 4,780,451 (issued Oct. 25, 1988) describes compositions and methods using LH and follicle stimulating hormone (FSH) to produce superovulation in cattle Farin et al. (1988 Biol. Reprod. 38:413-421) describes the effect on ovine luteal weight of intravenous administration of 300 IU of hCG on Days 5 and 7.5 of the estrus cycle, without insemination. Hoyer and Niswender (1985 Can. J. Physiol. Pharmacol. 63(3):240-248) describe the regulation of steroidogenesis in ovine luteal cells. Juengel and Niswender (1999 J. Reprod Fertil. Suppl. 54:193-205) describe the molecular regulation of luteal progesterone in domestic ruminants. U.S. Pat. No. 5,589,457 (issued Dec. 31, 1996) describes methods for synchronizing ovulation in cattle using GNRH, LH, and/or hCG and PGF2α.
The gonadotropins form a family of structurally related glycoprotein hormones. Members include chorionic gonadotropin (CG), follicle-stimulating hormone (FSH), luteinizing hormone (LH), also known as lutropin, and thyroid stimulating hormone (TSH). FSH, LH, and TSH are present in most vertebrate species and are synthesized by the pituitary gland. CG has only been found in primates, including humans, and in horses, and is synthesized by placental tissues. The gonadotropins are heterodimers of two subunits, α and β, which are associated with non-covalent bonds. Within a species, the α subunit is essentially identical for each member of the gonadotropin family. The β subunits are different for each member, but are similar in structure. The β subunit of hCG is substantially larger than the other β subunits, in that is contains an additional 34 amino acids at the C-terminal end of the protein.
The effects of LH depend on the sex of the organism. In sexually mature females, LH stimulates the follicle to secrete estrogen in the first half of the menstrual cycle. A surge of LH triggers the completion of meiosis I of the egg and release of the egg (ovulation) in the middle of the cycle stimulates the now-empty follicle to develop into the corpus luteum, which secretes progesterone during the latter half of the menstrual cycle. In males, LH acts on the interstitial cells of the testes stimulating them to synthesize and secrete the male sex hormone testosterone. LH in males is also known as interstitial cell stimulating hormone (ICSH).
Production of recombinant bovine LH is described in WO 90/02757 (published Mar. 22, 1990), U.S. Pat. No. 6,455,282 (issued Sep. 24, 2002); U.S. Pat. No. 5,639,639 (issued Jun. 17, 1997), U.S. Pat. No. 5,767,251 (issued Jun. 16, 1998), Nilson (1987) J. Reprod. Fertil. Suppl. 34:227-36, Boime et al. (1992) Seminars in Reprod. Endocrin. 10:45-50, and Kaetzel (1985) PNAS USA 82:7280-7283. A process for the purification of recombinant LH is described in WO 01/62774 (published Aug. 30, 2001). U.S. Pat. No. 5,929,028 (issued Jul. 27, 1999) describes liquid gonadotropin containing formulations that may include LH. Otieno et al. (2002 Reproduction 123(1):155-162) describes expression of LH genes in bovine conceptuses.
There is a need in the art for a safe therapeutic for maintaining pregnancy of post-inseminated cows.
All publications and patent applications cited herein are incorporated herein by reference in their entirety to the extent that they are not inconsistent with the disclosure herein. Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on subjective characterization of the information available to the applicant, and does not constitute any admission as to the accuracy of the dates or contents of these documents.